This invention relates to the field of fluorescent substrates for hydrolytic enzymes, and also relates to the field of biological assays.
In the field of biomolecular analysis, labeling with either radioactive isotopes or colorimetric dyes have historically been the detection methods of choice. However, due to the difficulties of working with radioactive isotopes and the limits of sensitivity of colored dyes, there is an increasing interest in the use of luminescent molecules for labeling and detection. In particular, there is increasing interest in developing luminescent detection systems that not only offer greater sensitivity than radioactive or colorimetric detection but also enable simultaneous detection of multiple analytes in the same sample.
Two types of luminescence are of general interest in biological assays: chemiluminescence and fluorescence. Chemiluminescence is the transient release of energy as light during a chemical reaction, which in most cases of biological interest involves a reaction catalyzed by a specific enzyme. Fluorescence is the emission of light induced by excitation of a molecule with light of a different wavelength. Unlike chemiluminescence, a fluorescent molecule can be derivatized so that it can be used either (i) to covalently attach an already fluorescent molecule as a label, or, (ii) as a xe2x80x9cfluorogenicxe2x80x9d enzyme substrate which can be converted, by action of a specific enzyme, into a fluorescent product which exhibits greatly enhanced fluorescence as compared to the starting substrate. In either case, detection relies upon the indirect detection of a specific enzyme by measurement of the rate or extent to which a substrate for that enzyme has been converted into a detectable chemiluminescent or fluorescent final product.
In biological assays, the activity of an enzyme is used to indirectly detect or measure the quantity of a complementary biological xe2x80x9ctarget.xe2x80x9d The enzymes most commonly used for this purpose are Alkaline Phosphatase, xcex2-Galactosidase, xcex2-Glucuronidase, xcex2-Glucosidase and Horse Radish Peroxidase. With the exception of HRP, the first four enzymes form a related set in that all four are hydrolytic enzymes that act on substrate molecules which have been derivatized at hydroxyl moieties to create the phosphoric acid, galactoside, glucoside and glucuronide substrates, respectively. The hydrolytic enzymes are widely used with calorimetric and chemiluminescent substrates and, despite the sensitivity limitations of current fluorogenic substrates, are also in widespread use, most commonly the substrates of 4-Methyl Umbelliferone (4MU) and Attophos. By contrast, although HRP is an important non-hydrolytic enzyme for both histochemistry and ELISAs using colorimetric and chemiluminescent substrates, fluorogenic peroxidase substrates are not extensively used for detection owing to their insufficient stability in aqueous solutions.
Numerous new chemiluminescent enzyme substrates for hydrolytic enzymes have been developed during the past decade as a result of which the advantages and limitations of chemiluminescent systems are now well known. Advantages include low non-specific chemiluminescence as the result of non-enzymatic hydrolysis, and, in the case of the 1,2-dioxetane xe2x80x9cglowxe2x80x9d reagents, a significantly greater sensitivity of detection as compared to calorimetric detection. Disadvantages of chemiluminescent detection include: laborious requirements for use, limitations on sensitivity arising from the transience of chemiluminescence itself, and, the broad spectral radiance of the chemiluminescent emission which precludes the simultaneous detection of multiple analytes in a single specimen.
Fluorescence detection is theoretically capable of circumventing the disadvantages of chemiluminescent detection, however, the former has long been regarded as less sensitive than chemiluminescence owing to (i) background light (xe2x80x9cstrayxe2x80x9d light) which exists in all fluorescence detectors because of the light source used to induce excitation, (ii) poor aqueous solubility of molecules which have a high fluorescence intensity, (iii) significant photoquenching and poor photostability in aqueous solutions, and, (iv) low rates of processing (turnover) of available fluorogenic substrates by their specific enzymes. Largely because of such limitations, fluorogenic hydrolytic enzyme substrates have been made available for biological research and clinical applications. The present invention addresses that need through the identification of novel molecular structures having physical properties which overcome the limitations of previous compounds.
Sato et al. ([1992] Chem. Pharm. Bull. 40(3):786-788) disclosed a new class of fluorogenic substrates for determination of acidic and alkaline phosphatases. They disclosed 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, xe2x80x9cpyraninexe2x80x9d) as a highly fluorescent compound in both acidic and alkaline environments. They also disclosed that the fluorescence intensity of pyranine is markedly quenched upon phosphorylation, and could be used as a substrate for the assay of acid and alkaline phosphatases and human serum phosphatases. However, the molecule disclosed by Sato et al. had a chemical composition consistent with the molecule being C16H6K5O13PS3 4H2O, and hence was not a halo-hydoxypyrene disulfonic acid (HHPDS).
Wolfbeis and Koller ([1983] Anal. Chem. 129:365-370) reported on the uses of esters of HPTS as fluorogenic substrates for esterases, however these authors neither disclosed nor suggested a halogenated variant of HPDS. Wolfbeis et al. ([1983] Anal. Chem. 314:119-124) characterized the fluorescence characteristics of HPTS as well as those of a number of other fluorescent indicators. These authors neither disclosed nor suggested a halogenated variant of hydroxypyrene compounds.
Koller ([1994] Amer. Biotech. Laboratory 13 (November):13-15) reported on the characteristics of 1-hydroxypyrene-3,6,8-tris(dimethylsulfonamide), as well as acetates, butyrates and other long-chain fatty esters, phosphate, sulfate, galactoside, glucoside, glucuronide, and N-acetyglucosaminide derivatives thereof. However, there was no disclosure or suggestion of a halo derivative of the hydroxypyrene compounds.
Tietze and Bayer ([1937] from the Wissenschaftl Hauptlaboratorium of I. G. Garbenindustrie-Werk Leverkunsen, Printed in Germany-Druck: Metzger and Wittig, Leipzig, pp. 189-210), provided an early analysis of the chemistry and fluorometric properties of pyrene sulfoacids and derivatives thereof. In the course of describing the manufacture of 3-aminopyrene-5,8,10-trisulfoacid, the preparation of 3-chloropyrene and sulfonation thereof to 3-chloropyrene-5,8,10-trisulfoacid was mentioned. However, there was no mention or suggestion of the fluorescence or lack thereof of this compound, nor was there any mention of phosphate or other derivatives of chlorodisulfoacid as fluorogenic substrates.
In U.S. Pat. Nos. 4,585,598 and 4,614,713, fluorogenic phosphate esters of hydroxy-pyrene-trisulfonic acids were disclosed for fluorometric determination of phosphatases. The disclosed water-soluble phosphoric esters were prepared by reacting the hydroxypyrene-trisulphonic acid with a phosphorus pentahalide followed by hydrolysis of the dihalogenophosphonyloxy compound. The patent neither discloses nor suggests the use of a monohalo-hydroxypyrene-disulfonic acid as a fluorogenic substrate.
In U.S. Pat. No. 5,132,432, a class of pyrenyloxy trisulfonic acids was disclosed and their utility in a number of biological and biochemical procedures was described. The disclosure of that patent is hereby incorporated by reference. While that patent mentions a wide variety of possible substituents for the pyrenyloxy trisulfonic ester compounds, there is no disclosure or suggestion of a halo-pyrenyloxy disulfonic acid derivative substituted with a phosphate, xe2x80x94O-galactoside, xe2x80x94O-glucoside, xe2x80x94O-glucuronide moiety or the use of such a compound as a fluorogenic substrate.
In U.S. Pat. No. 4,844,841, a class of pyrenetrisulfonic acids useful as fluorescent lipid probes was described. There was no disclosure or suggestion of the halo-pyrene-disulfonic acid derivatives of the present invention.
In U.S. Pat. No. 5,424,440, a class of benzothiazole compounds was disclosed as being water soluble fluorogenic substrates. While chemically unrelated to the present inventions, this patent is cited and incorporated herein for its review of the state of the art.
In WO 93/15097, pyrene-(1,3,6-trisulfonic acid)-8-D-glucuronide and like compounds were disclosed as being useful fluorogenic substrates for determination of glycohdyrolytic enzymes, such as -D-glucuronidase. This publication neither discloses nor suggests a halo-substituted pyrene-disulfonic acid fluorogenic substrate.
The subject invention provides a new class of fluorogenic substrates. In a specific embodiment, these substrates are the halo-pyrene-disulfonic acids and derivatives thereof. In contrast to the previously reported pyrene trisulfonic acid derivatives, these new substrates display very high rates of conversion to products when in the presence of an appropriate enzyme. Advantageously, upon enzyme hydrolysis, these compounds produce intensely fluorescent halo-pyrene derivatives which are useful as fluorescent markers in biological and biochemical systems. Equally important, these compounds are highly stable to non-enzymatic hydrolysis, thereby enabling assay applications providing great sensitivity and broad dynamic range.
Advantageously, the compounds of the subject invention have high solubility in aqueous media, longwave excitation and highly Stokes"" shifted emission wavelengths that can be used in fluorometric assays.
In a specific embodiment, the substrates of the subject invention can be represented by the structure (I): 
and salts thereof, wherein:
R1 is O, OH, phosphate, sulfonyl, sulfate, amine, sulfonamide, ketone, ester, or a substrate for a hydrolytic enzyme (including, for example, xe2x80x94O-galactoside, xe2x80x94O-glucoside, xe2x80x94O-glucuronide, and N-acetylglucosaminide); and
R2, R3, and R4 are, independently, xe2x80x94SO3, NR5R6, xe2x80x94SO2NR5R6, or a halide; wherein R5, and R6 are independently H, aryl, heteroaryl, heterocyclo, C1-6 alkyl, arylalkyl, heteroarylalkyl,heterocycloalkyl,or a monofunctional linker optionally linked to a detector molecule; provided that at least one of R2, R3, or R4 is a halide.
In a preferred embodiment, one of R2, R3, or R4 is a halide and the halide is chlorine.
In additional preferred embodiments, the substrates of the subject invention can have a formula represented by the structures (II), (III), (IV) or (V): 
and salts thereof, wherein:
R1, R2, R3, R4, R7, and R8, are, independently, O, OH, phosphate, sulfonyl, sulfate, amine, sulfonamide, ketone, ester, or a substrate for a hydrolytic enzyme (including, for example, xe2x80x94O-galactoside, xe2x80x94O-glucoside, xe2x80x94O-glucuronide, and N-acetylglucosaminide), xe2x80x94SO3, NR5R6, xe2x80x94SO2NR5R6, halide, or aryl group; wherein R5 and R6 are independently H, H, aryl, heteroaryl, heterocyclo, C1-6 alkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, monofunctional linker optionally linked to a detector molecule; provided that at least one of R1, R2, R3, R4, R7, or R8 is O, OH, phosphate, sulfonyl, sulfate, amine, sulfonamide, ketone, ester, or a substrate for a hydrolytic enzyme (including, for example, xe2x80x94O-galactoside, xe2x80x94O-glucoside, xe2x80x94O-glucuronide, and N-acetylglucosaminide).